Suppression of temperature sensitive mutations in oncogene-related CDC genes in Saccharomyces cerevisiae by catabolite repression resistance and cytoplasmic petite mutations

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.

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p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1212-1221.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1212-1221

Author(s):

R S Sikorski ◽

W A Michaud ◽

P Hieter

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle Progression ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Cycle Progression ◽

Tetratricopeptide Repeats ◽

Tetratricopeptide Repeat Protein ◽

Cdc Genes

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.

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TWO CELL DIVISION CYCLE MUTANTS OF SACCHAROMYCES CEREVISIAE ARE DEFECTIVE IN TRANSMISSION OF MITOCHONDRIA TO ZYGOTES

Genetics ◽

10.1093/genetics/102.1.9 ◽

1982 ◽

Vol 102 (1) ◽

pp. 9-17

Author(s):

Susan K Dutcher

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Nuclear Division ◽

The Other ◽

Temperature Sensitive ◽

Gene Products ◽

Wild Type ◽

Mitochondrial Movement ◽

Mitochondrial Transmission ◽

Cdc Genes

ABSTRACT Mutations in CDC genes of S. cerevisiae disrupt the cell cycle at specific stages. The experiments reported here demonstrate that two CDC genes, CDC5 and CDC27, are necessary for mitochondrial segregation as well as for nuclear division. The defect in the transmission of mitochondria was revealed by the examination of uninucleate and binucleate progeny of transient heterokaryons generated by using the kar1-1 mutation that disrupts nuclear fusion. One of the parents lacked mitochondrial DNA (?0) whereas the other parent had functional mitochondria (?+). When the parents of the heterokaryon were both wild-type (CDC), nearly all progeny received mitochondria at 21° and at 34°. Thirty-four of the 36 cdc mutations tested had no defect in transmission of mitochondria to zygotic progeny in crosses in which one parent was a cdc mutant and the other parent was not (CDC). However, the cdc5 and cdc27 mutations prevented the transmission of mitochondria to cdc progeny at 34° but not at 21°; CDC progeny received mitochondria at either temperature. This defect was observed in crosses of cdc5 or cdc27 by wild-type cells regardless of which parent donated mitochondria to the zygote. The defect in mitochondrial transmission cosegregated in meiotic tetrads with the defect in mitosis demonstrating that both are likely to be caused by the same temperature-sensitive mutation. These results indicate that the CDC5 and CDC27 gene products are essential in two motility-related processes: mitochondrial movement from the zygote to the progeny and in mitosis.—Furthermore, the results suggest that the function performed by the CDC5 and CDC27 gene products for mitochondrial transmission differ in some fundamental way from the function performed for mitosis. The function necessary for mitosis can be supplied to the cdc5 (or cdc27) nucleus by the CDC5 (or CDC27) nucleus in the same heterokaryon but the function necessary for mitochondrial transmission cannot. Perhaps the function needed for mitochondrial transmission must be performed in the cell cycle preceding the actual segregation of mitochondria whereas the function needed for nuclear segregation can be performed at the time that mitosis occurs.

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Requirement for Three Novel Protein Complexes in the Absence of the Sgs1 DNA Helicase in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.1.103 ◽

2001 ◽

Vol 157 (1) ◽

pp. 103-118 ◽

Cited By ~ 16

Author(s):

Janet R Mullen ◽

Vivek Kaliraman ◽

Samer S Ibrahim ◽

Steven J Brill

Keyword(s):

Saccharomyces Cerevisiae ◽

Genome Stability ◽

Protein Complexes ◽

Ring Finger ◽

Dna Helicase ◽

Open Reading Frames ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Dna Helicases ◽

Cell Extracts

Abstract The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability. To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed. We obtained mutations in six complementation groups that we refer to as SLX genes. Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species. None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase. Analysis of the null mutants identified a pair of genes in each of three phenotypic classes. Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1. Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex. Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology. The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts. The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts. We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.

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Deletion of QCR6, the gene encoding subunit six of the mitochondrial cytochrome bc1 complex, blocks maturation of cytochrome c1, and causes temperature-sensitive petite growth in Saccharomyces cerevisiae.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42253-8 ◽

1994 ◽

Vol 269 (2) ◽

pp. 1270-1275

Author(s):

M. Yang ◽

B.L. Trumpower

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytochrome Bc1 ◽

Temperature Sensitive ◽

Mitochondrial Cytochrome ◽

Bc1 Complex ◽

Cytochrome Bc1 Complex ◽

Gene Encoding ◽

Cytochrome C1

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Demarcation of a sequence involved in mediating catabolite repression of the gene for the 11 kDa subunit VIII of ubiquinol-cytochrome c oxidoreductase in Saccharomyces cerevisiae

Nucleic Acids Research ◽

10.1093/nar/16.13.5797 ◽

1988 ◽

Vol 16 (13) ◽

pp. 5797-5811 ◽

Cited By ~ 20

Author(s):

A.C. Maarse ◽

M. de Haan ◽

A. Bout ◽

L.A. Grivell

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytochrome C ◽

Catabolite Repression

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Temperature sensitive nop2 alleles defective in synthesis of 25S rRNA and large ribosomal subunits in Saccharomyces cerevisiae

Nucleic Acids Research ◽

10.1093/nar/29.14.2927 ◽

2001 ◽

Vol 29 (14) ◽

pp. 2927-2937 ◽

Cited By ~ 21

Author(s):

B. Hong

Keyword(s):

Saccharomyces Cerevisiae ◽

Temperature Sensitive ◽

Ribosomal Subunits

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Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.6891-6899.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 6891-6899 ◽

Cited By ~ 92

Author(s):

Xuan Wang ◽

Grzegorz Ira ◽

José Antonio Tercero ◽

Allyson M. Holmes ◽

John F. X. Diffley ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Synthesis ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

S Phase ◽

Temperature Sensitive ◽

Replication Proteins ◽

Dna Ligases ◽

Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

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SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2154 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2154-2164 ◽

Cited By ~ 50

Author(s):

D J DeMarini ◽

M Winey ◽

D Ursic ◽

F Webb ◽

M R Culbertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Gene Product ◽

Signal Sequence ◽

Null Alleles ◽

Nucleoside Triphosphate ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

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